Isolation of pure, intact RNA is a critical step for analysis of gene expression in molecular biology, clinical, and biotechnology applications. Methods of RNA isolation have been developed in an attempt to achieve this goal. The most frequently used methods for RNA isolation are based on phenol extraction, precipitation from chaotropic salt solutions, and adsorption on silica (Ausubel et al, 2002), reviewed in my U.S. Pat. Nos. 4,843,155; 5,346,994; and 5,945,515. The method described in the '155 patent is frequently referred to as the single-step method and extracts RNA with a phenol-guanidine solution at pH 4. Its effectiveness and simplicity make the single-step method the most frequently used method for isolating RNA.
An improvement of the single-step method, described in my subsequent '994 patent, allowed simultaneous isolation of RNA, DNA, and proteins from the same sample by phenol-guanidine extraction at pH 4-6. A biological sample is homogenized and the homogenate is subjected to phase separation using a hydrophobic organic solvent such as chloroform or bromochloropropane. Following centrifugation, the mixture separates into the top aqueous phase containing RNA, and the interphase, and organic phase containing DNA and proteins. The aqueous phase is collected and RNA is precipitated and washed with alcohol.
In the single-step method described in the '155 and '994 patents, a careful collection of the separated aqueous phase is critical for the quality of the isolated RNA. Small amounts of the interphase and organic phase can be easily removed together with the aqueous phase, which results in contamination of the isolated RNA with DNA and proteins. Also, collection of the aqueous phase requires a manual approach, which is an obstacle in adapting the single-step method for automation.
The reagents and methods described in the '155 and '994 patents provide substantially pure, undegraded RNA. However, RNA isolated according to the '155 and '994 patents contains a residual amount of genomic DNA, which can be detected by reverse transcription-polymerase chain reaction assay (RT-PCR). Thus, RNA isolated in accord with the '155 and '994 patents must be further purified to render it DNA-free (Guan at al, 2003; Girotti and Zingg, 2003). The contaminating genomic DNA serves as a matrix for DNA polymerase, yielding additional amplification products and distorting RNA-dependent RT-PCR. The DNA contamination in RT-PCR can be only partially alleviated by using a set of primers encompassing exon-intron sequences in the genomic DNA because the presence of pseudogenes, containing no introns, makes this approach unreliable (Mutimer 1998).
Modifications to the single-step method have improved the quality of the isolated RNA. In one modification, RT-PCR inhibitors were removed by adding a lithium chloride precipitation step (Puissant, 1990; Mathy, 1996). In another modification, alcohol precipitation of RNA in the presence of salt increased purity of the isolated RNA (Chomczynski, 1995). These modifications, however, were not effective in removing DNA contamination.
A common practice for removing contaminating DNA is to treat an RNA-containing sample with deoxyribonuclease (DNase). Following DNase treatment, the RNA-containing sample is extracted sequentially with phenol and chloroform. In an effort to limit DNA contamination, an additional DNA precipitation step was included in the single-step method. The contaminating DNA was precipitated from the aqueous phase by adding one-third the volume of 95%w/w ethanol (Siebert, 1993). The final concentration of ethanol was about 24%w/w. The author indicated that, at this low ethanol concentration, DNA was precipitated while RNA remained in solution. RNA was precipitated from the solution by adding additional alcohol. This protocol, however, yielded RNA that was still contaminated with DNA, evidenced as a visible band upon analyzing the isolated RNA on an agarose gel stained with ethidium bromide and by RT-PCR.
In another effort to diminish DNA contamination and improve the quality of RNA in the single-step method, Monstein (1995) in a laborious procedure increased the pH of the phenol extraction to pH 4.1-4.7 and treated the sample with proteinase K, followed by another round of phenol extraction, precipitation, and ethanol wash. Despite this prolonged procedure, DNase treatment was still necessary to obtain DNA-free RNA ready for use in RT-PCR.
Separating RNA from DNA was also achieved by phenol extraction at pH 4 without adding guanidine salts (Kedzierski, 1991). However, the absence of guanidine salts during the procedure made RNA susceptible to ribonuclease (RNase), thereby degrading the RNA. A later improvement of this protocol employed phenol extraction buffer at pH 4.2 in the presence of sodium dodecyl sulfate (Chattopadhyay et al., 1993). DNase treatment was also required in the RNA isolation method using a combination of the single-step method followed by the silica column procedure (Bonham, 1996). The use of this double purification protocol decreased DNA contamination, but the isolated RNA still contained genomic DNA that was detected by RT-PCR. Another method for isolating RNA used a monophase aqueous solution containing 10%w/w to 60%w/w phenol (U.S. patent application Publication 20030204077). In the absence of chaotropes, 15%w/w to 55%w/w monoalcohol, diol, or polyol was used to keep phenol in aqueous solution
Thus, a residual amount of DNA present in RNA isolated by the methods described in the '155 and '994 patents made it necessary to extend the procedure by including DNase treatment. This diminished the usefulness of the methods by prolonging procedures and unnecessarily exposing RNA to the possibility of degradation during DNase treatment and additional purification steps. However, removing residual DNA from RNA preparations is needed for RT-PCR based microarray determination of gene expression.
Previous methods for isolating RNA, as described in the '155 and '994 patents, were based on phenol extraction performed at pH 4 or higher. None of the previous modifications of the single-step method attempted to improve the quality of RNA by performing phenol extraction at a pH below 4. To the contrary, pH 4 as used in the first '155 patent was increased in the next '994 patent to a pH ranging from 4 to 6. Similarly, the protocol described by Monstein (1995) increased the pH of the phenol extraction to pH 4.7. Another elaborate attempt to improve the single-step method increased the pH of the guanidine-phenol extract to pH 5.2 (Suzuki, 2003).
An alternative to the single-step method of RNA isolation was disclosed in U.S. Pat. No. 5,973,137, using non-chaotropic acidic salts. However, the single-step phenol extraction method is still the most frequently used method for RNA isolation. A publication describing the single-step method (Chomczynski 1987) is the fourth most cited paper in the database of the American Chemical Society and Institute for Scientific Information, and the most cited paper published within the last twenty years (CAS 2003, American Chemical Society).
New methods to enhance purity of isolated RNA are thus desirable.